Method for dosing a biological or chemical sample

ABSTRACT

The invention relates to a method for assaying a biological or chemical sample comprising the following steps:
         illuminating the sample ( 10 ) by means of a light beam ( 17 ) from a source ( 11 ),   producing an image including the image of the beam ( 18 ) diffused by the sample ( 10 ),   analysing the image according to reference criteria,   extracting information specific to the light/sample beam interaction,   calculating the assay.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority based on International PatentApplication No. PCT/FR2004/050289, entitled “Method for Dosing aBiological or Chemical Sample” by Francois Perraut and EmmanuelleSchultz, which claims priority of French Application No. 0350270, filedon Jun. 27, 2003, and which was not published in English.

TECHNICAL FIELD

This invention relates to a method for assaying a biological or chemicalsample.

The field of the invention is in particular that of measuring theconcentration of fluorescent molecules called fluorochromes contained insolutions. Such molecules are used to assay the amount of a givenbiological species. The number of molecules of this biological speciesis thus related to the amount of fluorescent molecules. A measurement ofthe intensity emitted in the excitation of these fluorescent moleculesmakes it possible to deduce, by calibration of the measuring apparatusused, the number or concentration of biological molecules. Suchmeasurements are routinely used in biology, chemistry and physics.

In the following description, for the sake of simplification, theinvention is described in terms of this field of measuring thefluorescence of samples.

PRIOR ART

Many commercial apparatus, such as fluorimeters andspectro-fluorimeters, enable the fluorescence of a solution to bemeasured. These apparatuses make it possible to obtain a measurement ina chamber of which the shape is variable and depends upon theapplication.

Other apparatuses for measuring in solution use capillaries as achamber. These are, for example, measuring systems for anelectrophoresis apparatus.

In all of these apparatuses, a sample placed in a chamber, with which asingle detector is associated, is measured.

In some spectro-fluorimetry applications, multiple detectors are used.The fluorescent molecule emission spectrum is then dispersed over animage sensor so as to simultaneously measure the energy in all of thewavelengths, which amounts to the use of a single detector for eachspectral interval. A plurality of pixels are sometimes associated in theorthogonal direction with that of the dispersion so as to carry out aso-called “binning” operation which enables the signal-to-noise ratio ofeach spectral measurement to be increased while reducing the readoutnoise of the detectors with the photon flow. Multiple detectors are alsoused in some multi-sample apparatuses. The presence of a plurality ofsamples then requires the use of a plurality of chambers, and themeasurement for each sample is performed with an image sensor.

The detection sensitivity of such apparatuses is inadequate to assaysmall numbers of molecules, typically of the order of the picomole,either for diagnosing a disease or for studying the purity of asolution. Some assay types are even impossible to carry out below acertain concentration: in the field of immunoanalysis (antigen assay),the statistical detection threshold of the prior art, expressed in termsof target concentration, the lowest obtained for a measurement insolution is of the order of one hundred picomoles. Typically, commercialchamber apparatuses do not enable a fluorescence below a targetconcentration of 1 nM (nanomole) to be measured.

To reduce the detection limit, the light of a laser source can befocused into a very small space, as is done in capillaryelectrophoresis. The sample then passes into a capillary of a fewhundred micrometers of diameter. The detection limit obtained is of theorder of the nM, as described in reference document [1] at the end ofthe description.

Such a marker detection limit exists in a complex system comprisingintegrated confocal optics searching the interior of a capillary, asdescribed in reference document [2].

The measuring systems use capillaries not allowing for a very highsample rate, for example, of one dozen to one hundredmicrolitres/minute. It is therefore impossible to perform a measurementon a high-volume sample. The resulting selection reduces the molecularsampling and increases the detection threshold. For example, if it ispossible to detect the presence of a single molecule by fluorescencecorrelation techniques, as described in reference document [3], theprobe volume is very small, of the order of a femtolitre. Detecting amolecule in such a volume gives a detection limit of the order of a nM.

To increase the power density in the excited volume, the excitationlight can be focused. As the fluorescence emission is nearlyproportional to the amount of energy received in the excitation, theincrease in power density enables the number of photons emitted influorescence to be increased. For a well-designed measuring system, asis the case for most commercial instruments, the number of measuredphotons is greater and the uncertainty with regard to this measurementis lower. This uncertainty varies as 1/√{square root over (N)} where Nis the number of photons transformed into electrons during theconversion by the detector. This justifies the choice of an increase inpower density in the excited volume. However, a high power density isaccompanied by a photo-extinction, which is all the more rapid as thelight energy is high. For the measurement of a very low concentration offluorescent molecules, it is then necessary to expose this volume for anamount of time greater than the photobleaching time, which correspondsto a property of the fluorescent molecules consisting of stopping theemission of light at the end of a certain time period. It is thenimpossible to collect enough photons to reach the required detectionthreshold.

Factors that limit the detection threshold lie in the autofluorescenceof liquids, which is an intrinsic fluorescence of these media, and inthe Raman scattering. The level of emitted light indeed reducesdetection performance because the photoluminescence “offset” of thebuffer used is of the same nature as the signal, i.e. “specific”, to bedetected. If the measuring system is only limited by the “Schottkynoise”, also called “photon noise”, then the smallest statisticallymeasurable signal S_(min) is equal to 3× √{square root over (Offset)},where “Offset” is a measurement expressed in primary electrons(electrons resulting directly from the conversion of photons by aphotocathode in the case of a photomultiplier or a semiconductorsurface) and 3 is an arbitrary factor that guarantees 99% discriminationbetween S_(min) and Offset. To solve such a problem, the solutions ofthe prior art consist of:

1) a careful selection of liquids,

2) a selection of the marker,

3) an increase in the measuring time in order to accumulate photons,

4) an increase in the excitation power in order to collect more photons.

However, the main factor limiting the detection threshold is thenon-reproducibility of the measurements, which, for low signal levels,becomes dominant very quickly. This non-reproducibility essentiallyarises out of a poor mechanical repositioning of the measuring chamberand the light that is collected by the liquid meniscus in said chamberand which is randomly directed into the space.

A solution for reducing this type of non-reproducibility consists ofperforming the injection in the chamber of a greater volume of solution.However, such an injection is adequate for obtaining good sensitivity.Moreover, it is not always possible or desirable to work with largevolumes.

The mechanical positioning is not easy to improve. In addition, in sucha solution, variations caused, for example, by ambient lighting and thevariation in the form of the meniscus are not addressed.

Another solution for reducing this type of non-reproducibility consistsof using chambers including a transparent “black” glass window, whichcorresponds to the zone taken into consideration for the measurement.This solution, however, reduces the photon flow collected by themeasuring system, and therefore increases the detection limit. Moreover,it does not enable the “offset” variations to be known, which can becaused by a change in the ambient light, by poor surface conditions onthe sides of the chamber (soiling, scratches, and so on), or by adiffusion from the meniscus and the walls.

Thus, these various solutions of the prior art do not allow for goodsensitivity or good reproducibility of the measurement.

The aim of the invention is to overcome these disadvantages by proposinga new method for assaying a biological or chemical sample that uses adevice for spatial recording of the image of interaction between thelight coming from a source and this sample as a means for selecting thenecessary information.

DESCRIPTION OF THE INVENTION

The invention relates to a method for assaying a biological or chemicalsample, comprising the following steps:

-   -   optionally placing the sample in a chamber of which all of the        sides are transparent,    -   lighting the sample by means of a light beam from a source,

characterised in that it also comprises the following steps:

-   -   producing an image including the image of the light diffused by        the sample,    -   analysing the image according to reference criteria,    -   extracting information specific to the light/sample beam        interaction,    -   calculating the assay.

In this method, the diffusion can be Raman scattering, fluorescencescattering, molecular diffusion or particle scattering. The analysis canconsist of a study of the spatial structure of the image and of thedistribution of light energy in this image. The assay can be calculatedwith respect to a calibration between the measurement of light energyand the sample concentration or amount. The assay can also be calculatedwith respect to the analysis of the kinetics of the biological orchemical reaction.

Advantageously, in this method, a first zone of interest around theexcited volume zone, and a second zone of interest next to this firstzone are defined, and the specific signal is measured by carrying outthe subtraction between the sum of all of the pixels of the first zoneand the sum of all of the pixels of the second zone.

The invention has the following advantages:

-   -   It makes it possible to obtain a much lower experimental        detection limit than that of the conventional systems.    -   It makes it possible to perform an assay using a large volume of        solution with a high flow rate, and therefore to consider        applications such as the analysis of river waters and building        ventilation systems.    -   It does not require the light to be focused in a small volume.        The photobleaching of the fluorescent molecules is therefore        very low.    -   It makes it possible, due to the shape of the chamber, to        simultaneously excite a large number of molecules, which enables        numerous photons to be collected.    -   Neither the autofluorescence nor the Raman scattering of the        liquid medium limits the sensitivity of the invention. It        therefore makes it possible to work with all commercial markers,        thereby reducing the marking constraints.    -   The non-reproducibility due to the chamber mechanics, cleaning        or deterioration of the optical surfaces of the chamber, and the        presence of artefacts (bubbles, dust) are no longer a        constraint. The invention makes it possible, by image analysis,        to reduce and even eliminate these. A shift in the position of        the chamber in the measuring system of the invention or a shift        in the excitation light of the medium can be fully corrected        after measuring the position of the fluorescent trace in the        recorded image. Similarly, dust present in the excited volume,        which could significantly modify the measurement, is small with        regard to the excited volume, and can thus be identified and        removed from the measurement without losing the latter, which is        impossible to perform with a mono-detector. In the event of        deterioration of the optical surfaces of the chamber, which can        lead to a modification in the amount of light that actually        excites the sample, the invention enables the variations in the        effective power to be known and the measurement to be corrected.    -   The variations in ambient light caused either by the sample or        by the environment are compensated by a measurement of any        “offsets” in the image, then the removal thereof.    -   The analysis of the information in an image can be performed on        the basis of predetermined elements (shading function,        predetermined positions of the various useful zones), or        dynamically in order to deal with random and/or unforeseen        disturbances by applying image-processing methods (entropy        maximisation, neural network).    -   The dynamics of the assay of the invention, which, for a certain        type of measurements, makes it possible to experimentally        achieve biological assay dynamics of 2 200, is much higher than        that of the prior art, which, for the same type of measurements,        is typically between 5 and 10.

The invention can be applied to a number of fields, and, in particular:

-   -   in all of the fields where it is useful to measure a fluorescent        solution,    -   in biology, and more specifically for the assay of biological        molecules or molecules of biological interest: antigens,        antibodies, peptides, DNA, cells, bacteria, and so on, for a        clinical diagnosis,    -   in chemistry (assay),    -   in pharmacy: assay of activity, contamination, and so on,    -   in physics: search for product traces, fluidic traces, mixture        analysis, and so on.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a bottom view of a first embodiment of a deviceimplementing the method of the invention.

FIGS. 2 and 3 show an image of the chamber obtained with theimage-recording device shown in FIG. 1.

FIGS. 4 to 6 show a second embodiment of a device implementing themethod of the invention.

FIG. 7 shows a third embodiment of a device implementing the method ofthe invention.

FIGS. 8 to 10 show three examples of an embodiment of a deviceimplementing the method of the invention.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The method of the invention is a method for assaying (measuring aconcentration or an amount) of a biological sample or a sample ofbiological interest (antigens, antibodies, peptides, DNA, cells,bacteria, toxins) or a chemical sample (solvent, dissolved gas,preparation, chemical activity), which can be a solid, a liquid, a gel,and so on.

In a first embodiment shown in FIG. 1, a sample 10 is illuminated by alight beam 17 coming from a source 11 provided with the fluorochromeused. For example, a 488-nm argon laser can be used for fluorescein or ahelium-neon laser at 633 nm can be used for Cy5. The sample 10 is placedin a chamber 12, for example, with a rectangular cross-section, of whichall of the sides are transparent. The cross section of this chamber 12can indeed be rectangular, square, cylindrical or elliptical. Anobjective system 13, equipped with a blocking filter 14, is mounted infront of a device for recording the spatial structure of an image 15,for example, a CCD camera or a scanning system, connected to aprocessing component 16. The device 15 that receives the beam 18diffused by the sample 10 makes it possible to record an image fromwhich a specific measuring signal can be extracted.

The method of the invention includes the following steps:

-   -   illuminating the sample 10 by means of a light beam 17 coming        from the source 11, which can be a gas laser, a solid laser, a        laser diode, an electroluminescent diode, an organic diode, a        spectral bulb such as a halogen, mercury, xenon, or deuterium        bulb,    -   producing an image of the light beam 18 diffused by the sample        10, wherein the origin of the diffusion can be Raman scattering,        fluorescence scattering, molecular diffusion (Rayleigh        scattering) or particle scattering (use of nanoparticles),    -   analysing the image with respect to references, which analysis        consists of examining the spatial structure of the image and the        distribution of light energy in this image, wherein said        references can be constituted, for example, by the experience of        a user or by morphological criteria (form and position of a        light trace), photometric criteria (frequency of spatial        variations of the light in the image), or statistical criteria        (variation of measurement estimators, image entropy),    -   extracting information specific to the interaction between the        beam 17 and a sample 10, which extraction consists, for example,        of arithmetic operations between the image and other images or        constants (for example, subtractions, additions, divisions,        multiplications), morphological operations (erosion, dilation,        binarization, clipping, segmentations, offset correction) or        photometric operations (polynomial corrections, convolutions,        filters, thresholding),    -   calculating the assay with respect to a calibration between the        measurement of light energy and the concentration or quality of        the biological or chemical sample. This calculation can also be        performed by recording the kinetics of the biological or        chemical reaction and by analysing this kinetic using methods        known to a person skilled in the art.

In the method of the invention, the measurement is performed in an imageobtained by the device for recording the spatial structure of an image15. The invention does not lie in the use of such a device 15, butprimarily in:

-   -   the recording of the beam 18 diffused by the sample 10 in the        form of an image,    -   the extraction of information from this image,    -   the adaptive side obtained by the application of an image        analysis.

FIG. 2 shows the image of the chamber 12 obtained with the device forrecording the spatial structure of an image 15. The chamber can havesmaller dimensions than those of the image. It can, for example, bereplaced by one or more capillaries.

-   -   In addition, the light beam can be either smaller or larger than        the chamber.

In this image, a plurality of zones can be distinguished:

-   -   an illuminated volume zone 20, which corresponds to the volume        of the chamber 12 excited by the beam 17,    -   the zone 21 where said beam 17 enters the chamber 12,    -   the zone 22 where said beam 17 exits the chamber 12,    -   a meniscus zone 23,    -   an artefact zone 24.

Thus, as shown in FIG. 3, it is possible to define a first region ofinterest 25 around the illuminated volume zone 20 and a second region ofinterest 26 next to this zone 25. The measurement of the specific signalis then given by the calculation: ΣRI₁-ΣRI₂; that is, the subtractionbetween the sum of all of the pixels of the first region of interest 25and the sum of all of the pixels of the second region of interest 26.

In FIG. 3, the two regions of interest 25 and 26 have the same size,which is not essential. When these regions do not have the same size, itis simply necessary either to average the grey levels of each region, orto balance the values by the number of pixels.

The image analysis thus represented leads to certain observations:

-   -   The fluorescent trace of the light beam (zone 20) gives the        specific signal.    -   The meniscus (zone 23), delimiting the liquid from the air, has        a strong light. It originates from said trace. The form of this        meniscus is highly random. The amplitude of the signal coming        from this meniscus is therefore highly variable.    -   An artefact (zone 24) can be, for a given assembly, a spring        lock washer designed to ensure the proper mechanical positioning        of the chamber 12.

The zones 21 and 22 correspond respectively to the points of entry andexit of the light beam in the chamber 12.

With another adjustment of the display thresholds, it is easier to showthe lowest light levels.

The method of the invention makes it possible to improve the variationcoefficient (CV) (standard deviation/mean). Indeed, the coefficient CVobtained with the method of the invention is lower than the coefficientCV calculating by obtaining the sum of all of the pixels of a CCDcamera, which corresponds to a measurement performed with amono-detector. The coefficient CV obtained with the method of theinvention is of the same order of magnitude as that obtained with a highsolution volume, as considered previously in the introduction to saidapplication. This coefficient CV obtained with the method of theinvention is lower than that obtained by the measurement in each of thezones of interest 25 and 26. The subtraction of the measurementsperformed in these two zones of interest makes it possible to correctlighting variations that affect all of the regions. The invention thusmakes it possible to perform spatial filtering in the plane that enablesthe signal containing specific information on the fluorescence to beextracted. Moreover, the invention makes it possible to extract theregions of interest which are truly relevant in an image or apseudo-image, while a measuring system using a single mono-pixeldetector cannot perform such a function.

In a second embodiment, a mono-detector associated with a matrix ofpixels with programmable transparency 30 such as a liquid crystal ormicro-mirror matrix or any other equivalent system is placed in front ofthe chamber 12 shown in FIG. 4. This matrix 30 is intercalated betweenthe chamber 12 and the detector via a system for forming images or not.A first measurement is then performed by “opening” the pixelscorresponding to the first zone of interest 25, as shown in FIG. 5, thena second measurement is performed by opening the pixels corresponding tothe second zone of interest 26, as shown in FIG. 6.

The use of such a matrix 6 with variable transparency makes it possibleto avoid the systematic recording of an image, for example, byperforming the following steps:

-   -   recording the image of the beam diffused by successively        opening/closing all of the pixels of the matrix 30 in        synchronisation with the measurement carried out by the        mono-detector,    -   analysing the image and defining the zone(s) of interest        enabling the specific information to be extracted,    -   recording such parameters for a subsequent use,    -   in the analysis of a given sample, performing successive        openings of regions defined in the analysis step and recording        the results of the measurement for each of these zones,    -   extracting the necessary information,    -   calculating the assay.

In a third embodiment shown in FIG. 7, two mono-pixel detectors 35 and36 each observe a region of interest 25 or 26. Two image formation means37 and 38 are placed respectively in front of each of these twodetectors 35 and 36. The measurement of the signal coming from the firstregion of interest 25 is performed with detector 35, and that for thesecond region of interest 26 is performed with detector 36. The zone 39shows the front view of the fluorescent trace.

The invention makes it possible to adapt the extraction of the specificsignal to experimental conditions. For example, if the chamber has movedbetween two series of measurements, it is possible, by an automaticanalysis of the image, to automatically reposition the regions ofinterest, an operation that could not be performed with a static system.

EXAMPLES OF EMBODIMENTS OF THE INVENTION

The three examples of embodiments described below correspondrespectively to the three embodiments defined above.

In a first example of an embodiment shown in FIG. 8, a light source 40,for example a laser or an electroluminescent diode, excites, in achamber 41, the liquid containing fluorescent molecules through opticswith a configuration that is not shown, and various accessories such asa shutter 42 and a diaphragm 43. A first objective 44, placed, forexample, perpendicularly with respect to the primary direction of thelight beam, collects a portion of this light beam, emitted byfluorescent molecules in the chamber 41. A blocking filter 45 is placedbehind the first objective 44, just in front of a second objective 46.The association of objectives 44 and 46 enables an image of the chamber41 to be formed on the image detector 47 which is connected to a commandand control system 49. For a chamber 41 with an internal width of 1 cm,this detector 47 can be a detector of 512×512 pixels with a side of 10μm. The first objective 44 can have a focal length of 50 mm and thesecond objective 46 can have a focal length of 25 mm.

In a second example of an embodiment, shown in FIG. 9, of which theconfiguration is the same as that of FIG. 8 for excitation, only onedetector is used. A matrix 56 with variable transparency, which can be aliquid crystal matrix or a micromirror matrix, acts as a fielddiaphragm. The matrix 56 can be replaced by a mobile window actuated bya mechanical or electromechanical actuator, for example an electromagnetor an electric motor.

In a third example of an embodiment shown in FIG. 10, the configurationfor exciting the inside of the chamber 41 is the same as in FIG. 8, andthe configuration for the light collection is the same as that in FIG.9. Two mono-detectors 50 and 51, for example, offset photomultipliers,enable two different regions of the chamber 41 to be observed. In frontof each detector 50 and 51, recapture optics 52 and 53 enable the imageof the region of interest to be formed on a field diaphragm 54 and 55,which limits the region observed. A blocking filter can be placed infront of, in or behind the diaphragm. The zone 56 represents thefluorescent trace.

REFERENCES

[1] “Some applications of near-ultraviolet laser-induced fluorescencedetection in nanomolar- and subnanomolar-range high-performance liquidchromatography or micro-high performance liquid chromatography” by N.Siméon, R. Myers, C. Bayle, M. Nertz, J. K. Stewart, F. Couderc (2001,Journal of Chromatography A, Vol 913, I 1-2, pages 253-259).

[2] “Performance of an integrated microoptical system for fluorescencedetection in microfluidic systems” by J. C. Roulet, R. Volkel, H. P.Herzig, E. Verpoorte, N. F. Rooij, R. Dandliker (2002, AnalyticalChemistry, Vol. 74 (14), pages 3400-3407).

[3] “Single molecule detection of specific nucleic acid sequences inunamplified genomic DNA” by A. Castro and J. G. Williams (1997,Analytical Chemistry, Vol. 69 (19), pages 3915-3920).

1. A method of assaying a biological or chemical sample, whichcomprises: placing said sample in a chamber having transparent sides;illuminating the sample using a light beam coming from a source;producing an image of the light beam diffused by the sample, whereinsaid image comprises an illuminated volume zone, a light beam point ofentry zone, a light beam point of exit zone, a meniscus zone, and anartefact zone; recording the spatial structure of the image; examiningthe spatial structure of the image and distribution of light energy inthe image with respect to one or more references, measuring noisecreated by the step of illuminating the sample, and defining one or moreregions of interest so that measuring information can be extracted;extracting the measuring information, wherein the extracted informationis specific to the interaction of the light beam with the sample;recording the measuring information; and calculating the assay withrespect to the measuring information.
 2. A method as recited in claim 1,wherein the diffusion is Raman scattering, fluorescence scattering,molecular diffusion or particle scattering.
 3. A method as recited inclaim 1, wherein the assay is calculated with respect to a calibrationbetween the light energy measurement and the sample concentration oramount.
 4. A method as recited in claim 1, wherein the assay iscalculated with respect to an analysis of the kinetics of the biologicalor chemical reaction.
 5. A method as recited in claim 1, wherein a firstzone of interest around the illuminated volume zone, which correspondsto the volume of the chamber excited by the light beam, and a secondregion of interest next to this first region are defined, and whereinthe measuring information is obtained by subtracting the sum of thesignals of all of the pixels of the first region of interest from thesum of the signals of all of the pixels of the second region ofinterest.
 6. A method as recited in claim 1, further comprising derivingthe concentration of fluorescent molecules contained in a solution.